Role of tumor suppressor p53 domains in selective binding to supercoiled DNA

Abstract

We showed previously that bacterially expressed full-length human wild-type p53b(1-393) binds selectively to supercoiled (sc)DNA in sc/linear DNA competition experiments, a process we termed supercoil-selective (SCS) binding. Using p53 deletion mutants and pBluescript scDNA (lacking the p53 recognition sequence) at native superhelix density we demonstrate here that the p53 C-terminal domain (amino acids 347-382) and a p53 oligomeric state are important for SCS binding. Monomeric p53(361-393) protein (lacking the p53 tetramerization domain, amino acids 325-356) did not exhibit SCS binding while both dimeric mutant p53(319- 393)L344A and fusion protein GCN4-p53(347-393) were effective in SCS binding. Supershifting of p53(320-393)-scDNA complexes with monoclonal antibodies revealed that the amino acid region 375-378, constituting the epitope of the Bp53-10.1 antibody, plays a role in binding of the p53(320-393) protein to scDNA. Using electron microscopy we observed p53-scDNA nucleoprotein filaments produced by all the C-terminal proteins that displayed SCS binding in the gel electrophoresis experiments; no filaments formed with the monomeric p53(361- 393) protein. We propose a model according to which two DNA duplexes are compacted into p53-scDNA filaments and discuss a role for filament formation in recombination.

Enhancement of p53 sequence-specific binding by DNA supercoiling

“…It appears probable that it is the core domain that recognizes the sequence, while the C-terminus strengthens this binding by interacting with the local supercoilstabilized structure. We have previously showed the involvement of the C-terminus in p53 binding to scDNA not containing the p53 target (Palecek et al, 1997;Brazdova et al, 2002). Here, we show that the C-terminus can also act as a positive regulator in p53 sequence-specific binding to scDNA.…”

“…Considering the local level of DNA supercoiling in eucaryotic DNA as an extremely dynamic system (van Holde and Zlatanova, 1994;Pearson et al, 1996) and the relations between the p53 and DNA topoisomerases as well as the results of this and earlier papers on p53 binding to scDNA (Palecek et al, 1997(Palecek et al, , 2001Kim et al, 1999;Mazur et al, 1999;Brazda et al, 2000;Brazdova et al, 2002;Jagelska et al, 2002), we propose new aspects of the regulation of p53 SSDB. We predict that, in the complex p53-regulatory network, p53 DNA binding is determined not only by properties of p53 (potentially resulting from postsynthetic modification and protein-protein interactions), but also by various properties of DNA, including local DNA superhelix density and the abilities of the given p53 target and its flanking sequences to undergo conformational changes.…”

“…The effect of DNA supercoiling in regulating the p53 biological activity and the SSDB has become a topic of interest only recently (Kim et al, 1997;Mazur et al, Palecek et al, 1997Palecek et al, , 1999Brazda et al, 2000;Brazdova et al, 2002;Jagelska et al, 2002) (for a review, see Hupp, 1999). In contrast to the prokaryotic genome, the eucaryotic genome was long believed to not be under superhelical stress due to accommodation of DNA writhing around histone octamers in nucleosomes (van Holde and Zlatanova, 1994;Pearson et al, 1996).…”

“…A molecular weight of 53 kDa of this post-translationally modified protein was considered in all calculations. The p53 core domain (fragment 94-312, p53CD) and p53 C-terminal deletion mutant (fragment 1-363, p53CD30) were expressed in bacteria, isolated and characterized as described (Brazdova et al, 2002).…”

“…p53 can modulate the activity of topoisomerases either by direct molecular interactions or by transcriptional regulation, suggesting a possible relation between the p53 function and the DNA topology. Wild-type p53 protein preferentially binds to scDNA even in absence of the target sequence in the DNA molecule (Palecek et al, 1997Brazda et al, 2000;Pospisilova et al, 2000;Brazdova et al, 2002). Strong binding of wt p53 to scDNA not containing the CON (e.g.…”